Hydrogen peroxide induces La cytoplasmic shuttling and increases hepatitis C virus internal ribosome entry site-dependent translation

Abstract

We have previously shown that physio/pathological levels of hydrogen peroxide (H ₂O ₂) stimulate translation from the hepatitis C virus (HCV) internal ribosome entry site (IRES) element in tissue-cultured cells. Here, using in vitro translation, we further show that H ₂O ₂ upregulates HCV IRES-dependent mRNA translation and correlates with an increase in intracellular oxidant level. Using Western blotting, immunocytochemistry, microscopy and affinity pulldown, we show that H ₂O ₂ stimulates HCV IRES-dependent translation and correlates with nuclear–cytoplasmic shuttling of the La autoantigen, resulting in enhanced binding of cytoplasmic La to HCV IRES RNA. The role of the La protein in H ₂O ₂-stimulated IRES-dependent translation is further confirmed by the ability of an anti-La antibody to suppress H ₂O ₂-activated IRES-dependent translation in vitro. This is further supported by the ability of an ectopically expressed dominant, negative La mutant protein to suppress H ₂O ₂-inducible IRES-mediated translation in Huh7 cells, transiently transfected with a bicistronic reporter and in a sub-genomic replicon cell line resembling a persistent infection. On the other hand, translation from the encephalomyocarditis virus IRES is diminished in the presence of H ₂O ₂, suggesting that H ₂O ₂ translational responsiveness is a specific property of the HCV IRES and is not a general phenomenon for all viral IRESs. Altogether, these results suggest that HCV adapts to physio/pathological oxidative stress in the host cell by mediating La cytoplasmic shuttling to enhance its IRES-dependent translation.